Bacteria are social organisms that display distinct phenotypes when present in groups, changes that often result in heightened virulence. Phenotypic responses to increased population size include formation of antibiotic-resistant sessile biofilm communities, modification of gene expression patterns in response to diffusible signals (quorum sensing), and group motility (swarming). Our understanding of bacterial social responses derives primarily from in vitro studies of bacteria in flasks and on culture plates, communities that often contain greater than 108 cells. Although the behavior of very large populations may be important in some environments, bacteria in nature often reside in dense micro-clusters having far fewer individuals; importantly, it has been proposed that these clusters are the primary means of transmission of many pathogens, including Pseudomonas aeruginosa, Staphylococcus aureus, and Vibrio cholerae. Despite the strong imperative to understand onset and maintenance of social phenotypes within bacterial micro-clusters, fundamental requirements for group behaviors in these populations remain largely unknown due to a dearth of technologies for organizing bacteria into 3D patterns that contain specified numbers of cells at tunable densities. In this proposal, we describe strategies for exploiting three-dimensional (3D) protein-based microfabrication to control, in some cases, dynamically, spatial arrangements of small bacterial populations, technology that will be used to elucidate the basis for population-dependent antibiotic resistance in bacterial micro-clusters. The studies in this R21 grant focus on the clinically important, Gram-negative bacterium P. aeruginosa, a primary cause of cystic fibrosis mortality, and will be extended in future studies to other pathogens, including S. aureus. PUBLIC HEALTH RELEVANCE: Many infections are caused by small aggregate populations of bacteria. These aggregate populations display important traits from a public health standpoint, including resistance to antimicrobials and high infectivity. The goal of this research proposal is to understand the molecular basis for these traits with the ultimate goal of devising new therapeutic strategies for treating them. .